In recent years, not only interesting similarities, but also striking differences between protein transport pathways in eubacteria, archaea, eukaryotes, and eukaryotic organelles have been documented (Pohlschröder et al., Cell 91:563-566 [1997]; Dalbey and Robinson, Trends Biochem. Sci., 24:17-22 [1999]; and Dalbey and Kuhn, Ann. Rev. Cell. Dev. Biol., 16:51-87 [2000]). Insights into the extent of conservation and divergence in these pathways were provided due to the availability of many complete genome sequences. Unfortunately, the biological significance of such insights has often proven difficult, to test as the majority of organisms with sequenced genomes are poorly amenable to biochemical or genetic approaches. In this respect, the Gram-positive eubacterium Bacillus subtilis, the complete genome sequence of which was published by Kunst et al. (Kunst et al., Nature 390:249-356 [1997]), has been a very useful exception, due to this organism's natural system for genetic transformation and its large capacity for the secretion of proteins directly into the growth medium (See, Tjalsma et al., Microbiol. Mol. Biol. Rev., 64:515-547 [2000a]). Nonetheless, much remains unknown regarding the biosynthetic pathways of Bacillus. 
The functional genomic approach to dissect the protein secretion process in B. subtilis has yielded a number of remarkable surprises. These surprises include striking differences in the composition of the general secretion (Sec) and twin-arginine translocation (Tat) pathways for the transport of secretory pre-proteins across the membranes of B. subtilis and E. coli (Bolhuis et al., J. Biol. Chem., 273:21217-21224 [1998]; Bolhuis et al., J. Biol. Chem., 274:24531-24538 [1999a]; Jongbloed et al., J. Biol. Chem., 275:41350-41357 [2000]; Robinson and Bolhuis, Nat. Rev. Mol. Cell. Biol., 2:350-356 [2001]; and van Wely et al., Microbiol., 146:2573-2581 [2000]).
In contrast to Escherichia coil (See, Fekkes and Driessen, Microbiol. Mol. Biol. Rev., 63:161-173 [1999]), the Sec-dependent translocation machinery of B. subtilis lacks a SecB component (van Wely et al., J. Bacteriol., 181:1786-1792 [2000]). Moreover, the B. subtilis SecDF component, which is present as a natural fusion protein, is merely required to optimize the efficiency of protein translocation under conditions of protein hyper-secretion at gram per litre levels (Bolhuis et al., [1998], supra), while the separate SecD and SecF proteins of E. coli are very important both for protein export and cell viability (Pogliano and Beckwith, EMBO J., 13:554-561 [1994]). In addition, in contrast to the twin-arginine translocation (Tat) machinery of E. coli that consists of the unique TatB and TatC components and the paralogous TatA and TatE components (See e.g., Robinson and Bolhuis, Nat. Rev. Mol. Cell. Biol., 2:350-356 [2001]), the Tat machinery of B. subtilis lacks distinguishable TatA/E and TatB components, while two paralogous TatC proteins with distinct functions are present (Jongbloed et al., [2000], supra).
Translocated pre-proteins of B. subtilis with Sec-type or twin-arginine signal peptides have been shown to be subject to processing by the largest number of type I signal peptidases (SPases) known in various organisms. In addition to five chromosomally-encoded SPases (SipS, SipT, SipU, SipV, and SipW [Tjalsma et al., Genes Dev., 12:2318-2331 (1998)]), some B. subtilis strains contain plasmid-encoded SPases (Meijer et al., Mol. Microbiol., 17:621-631 [1995]). Furthermore, SipW was the first known eubacterial SPase of a type that is mainly encountered in archaea and the eukaryotic endoplasmic reticular membrane (Tjalsma et al., [1998], supra; and Tjalsma et al., J. Biol. Chem., 275:25102-25108 [2000b]).
Another finding was that the unique type II SPase (Lsp) of B. subtilis (Prágai et al., Microbiol., 143:1327-1333 [1997]), which specifically catalyzes the maturation of lipid-modified pre-proteins is required for the secretion of non-lipoproteins, such as α-amylase, chitosanase, and lipase (Tjalsma et al., J. Biol. Chem., 274:1698-1707 [1999]; and Antelmann et al., Genome Res., 11:1484-1502 [2001]). When the negative effect of an Isp mutation on non-lipoprotein secretion was first observed for the α-amylase AmyQ, it was largely attributed to a possible malfunctioning of the lipoprotein PrsA, which is essential for the proper folding of various translocated proteins, such as AmyQ.
In addition, the original view that Gram-positive eubacteria would lack thiol-disulfide oxidoreductases for the formation of disulfide bonds in secretory proteins was disproved with the identification of three Bdb proteins (Bolhuis et al., J. Biol. Chem., 274:24531-24538 [1999b]). Indeed, any lesson learned regarding protein secretion and the relative contribution of homologs from Gram-negative bacteria is not necessarily be relevant to Gram-positive microorganisms. Thus, there remains a need in the art to provide means to assess and identify secretory proteins in Gram-positive organisms such as Bacillus. 